Core promoter sequences analyzed by PBD Langevin dynamics simulations.

<p>Experimentally verified transcriptional start sites (TSS) are shown in large letters. Common promoter sequence elements are indicated by colored boxes. For illustrative purposes, sequences that fit the element definitions but are not properly positioned relative to the TSS are also shown as colored letters. Deviations from the consensus sequence are indicated in gray. The sequences were obtained from the Eukaryotic Promoter Database (EPD, <a href="http://www.epd.isb-sib.ch/" target="_blank">http://www.epd.isb-sib.ch/</a>). The identity of each promoter is described in column 1, the sequence is shown in column 2, and the mode of regulation in column 3.</p

Collective opening profiles of the collagen nonpromoter sequence calculated from the PBD Langevin dynamic simulations.

<p>(A) Probability for collective opening (vertical axis) of ten base pairs starting at specific nucleotide position within the collagen intron (horizontal axis), as a function of bubble amplitude [Å]. For comparison the profile of the collagen promoter is also presented (bottom panel). Probability values are colored to the same scale between the promoter and the intron sequences, as shown below the plots. Nucleotide positions in the collagen promoter are labeled relative to the TSS (+1). The sequence identity is shown at the top. (B) Probability for opening (vertical axis) of amplitude threshold (tr)≥1 Å, starting at specific nucleotide positions (horizontal axis), as a function of bubble length [bp]. Probability values are colored to the same scale, as shown below the plots. The sequence identity is shown at the top. (C) Average lifetimes of DNA collective openings of amplitude tr≥1 Å (vertical axis), starting at specific nucleotide positions (horizontal axis), as a function of length [bp]. The average lifetimes of collective openings for the collagen promoter are shown below. The TSS is marked with a vertical line. The color scale shown below the plots represents the average lifetimes [ps].</p

Probability for DNA collective openings of mammalian core promoters, calculated from PBD Langevin dynamic simulations.

<p>The probability was determined from the lifetimes of all open states above a given length and amplitude, normalized over the time of the simulation (Eq. 2). (A) Probability for opening (vertical axis) starting at specific nucleotide positions (horizontal axis), as a function of bubble length [bp]. Probability values are colored to the same scale between promoters for comparison. Nucleotide positions are labeled relative to the TSS (+1). Promoter identity and bubble amplitude thresholds are shown at the top. The thresholds are chosen individually for each promoter, as the smallest values for which the TSS region begins to exhibit maximum probability. (B) Probability for opening (vertical axis) starting at specific nucleotide positions (horizontal axis), as a function of bubble amplitude [Å]. Probability values are colored to the same scale between promoters for comparison. Nucleotide positions are labeled relative to the TSS (+1). Promoter identity and bubble length thresholds are shown at the top of the panels. The thresholds are chosen individually for each promoter, as the smallest values for which the TSS region begins to exhibit maximum probability.</p

Average lifetimes of DNA collective openings of core promoter sequences, as a function of length[bp].

<p>For clarity, the same promoter profiles are shown from a different angle in the panels at the right. Nucleotide positions are shown relative to the TSS (+1). The TSS is marked with a vertical line. The color scale represents the average lifetimes [ps]. The identity of the promoters is shown above the panels.</p

The TRS expansion has an effect on the DNA bubble spectrum.

<p>EPBD based LMD simulations have been conducted on the: a) (CAG.CTG)₄₅ repeats and healthy (CAG.CTG)₁₀ repeats with 30 bp flanking huntington gene sequence; b) (GAA.TTC)₁₂₀ and (GAA.TTC)₆ MRS that are embedded in 50 bp frataxin gene sequence; c) (CGG.GCC)₂₄₀ and (CGG.GCC)₂₀ repeats together with 50 bp FMR1 gene flanking sequence. The y-axis represents the length of the bubbles in bp; the x-axis represents the number of the base pairs; the color axis gives the bubble duration in psec. The brackets above the panels denote the repeated sequence; red arrows- the largest and long-lived base-pairs openings.</p

Accumulation of (GAA.TTC) repeats leads to changes in local DNA breathing.

<p>BAD criteria are used to describe and compare the local base pair breathing of DNA sequences with different numbers of (GAA.TTC) repeats embedded within the frataxin gene [B7] promoter sequence. a) BAD coordinates [Å] are calculated with EPBD based MCMC simulations for sequence inserts with different numbers of repeats: (GAA.TTC)₆-black line, (GAA.TTC)₄₅-red line, and (GAA.TTC)₁₂₀-blue line. The position of the flanking sequence (fl) is shown above the panel. b) BAD coordinates for a randomized sequence with the same number of base pairs and G+C content as the (GAA.TTC)₄₁ sequence. The random sequence (red line) is missing the synchronized average base pair openings behavior of the symmetric (GAA.TTC)₄₁ (blue line). The nucleotide position is shown on the horizontal. The BAD coordinates are shown on the vertical in [Å].</p

Fis-DNA points-of-contact and inclusion/exclusion rules.

<p>(A) Qualitative depiction of the sequence logo for a palindromic Fis binding site, emphasizing inclusion rules (above the numbers indicating the locations in the binding segment) and exclusion rules (below the numbers). The rules were derived from previous studies (see the main text). Yellow indicates direct points-of-contact or positions of inclusion/exclusion rules; blue indicates location of the Fis bubble formation region. The colors of the nucleic acids are chosen as the commonly used ones in consensus sequence logos. (B) Crystal structure example of Fis-DNA binding complex visualized by the data from PDB code 3IV5 as submitted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002881#pcbi.1002881-Stella1" target="_blank">[51]</a>. The nucleotides (direct points-of-contact) participating in the inclusion/exclusion rules are labeled and highlighted in yellow.</p

Fis binding site modifications, Langevin dynamics simulations of DNA breathing, and EMSA experiments, the second set.

<p>(A) Langevin dynamics simulations reinforcing the local DNA breathing dynamics in FIS2^m3 via three O6-methylguanine modifications in the bubble formation region of FIS2 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002881#pcbi-1002881-t001" target="_blank">Table 1</a>). The direct points-of-contacts remain unchanged. (B) Polyacrylamide gel electrophoresis of dsDNA oligonucleotides sequences - FIS2, FIS2^m2, and FIS2^m3 - demonstrating gel migratory effects due to possible bubble formation (gel at 15%). (C) Langevin dynamics simulations demonstrating local disruption of the hydrogen bonds in the super-enhanced DNA local openings of the FIS1–FIS2 sequence (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002881#pcbi-1002881-t001" target="_blank">Table 1</a>) caused by the presence of five mismatches at the FIS1 bubble formation region. (D) EMSA demonstrating the lack in complex formation in FIS1–FIS2. Concentration of the FIS1 and FIS1–FIS2 oligomers were constant at 100 nM and Fis protein ranged from 0 to 0.75 µM. Sonicated salmon sperm DNA at 0.5–1 µg/µl was added to the binding reactions to eliminate non-specific binding. In Langevin dynamics simulations (panels A and C) the probability of bubble openings is represented by the same color map as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002881#pcbi-1002881-g005" target="_blank">Figure 5</a>; red denotes high probability and blue denotes low probability of opening. The probability is determined from the lifetimes of all open states with a given length (bp) and above amplitude of 1.0 (Å), normalized over the complete time of the simulation. The length of the transient bubbles, given in base pairs [bp], is shown along the vertical axis. The horizontal axis depicts base pair position; the bubble formation region is highlighted in blue while the points-of-contact are highlighted in yellow. The names of each of the sequences are shown in the panels while the complete nucleotide sequences could be found in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002881#pcbi-1002881-t001" target="_blank">Table 1</a>.</p

DNA oligomers investigated with EPBD Langevin dynamics.

<p>Bold capital letters indicate direct points-of-contact or positions of inclusion/exclusion rules, which are located at: <b>−7;−4;−3</b>, and <b>+7;+4+3</b> positions; the location of the Fis bubble formation region is between −2 and +2 positions; low case letters indicate base pair substitutions; italic capital letters indicate complementary strand mismatches; ^mG represents O6-methylguanine.</p

Correlation between Fis binding affinity and the generalized opening profile.

<p>(A) The MCMC average opening profiles (vertical axis in [Å]) of FIS1 and FIS2 as a function of the nucleotide position. The bubble formation region is highlighted in blue while the points-of-contact are highlighted in yellow. (B) Characteristic MCMC opening profile (COP) obtained as the average of the profiles (black squares) of the oligomers with in vitro Fis-DNA binding affinity with KD<1 nM (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002881#pcbi.1002881.s001" target="_blank">Table S1</a>). The red line represents a polynomial fit to the data. The bubble formation region is highlighted in blue while the points-of-contact are highlighted in yellow. In panels (A) and (B), the horizontal axes indicate base pair position whereas the vertical axes indicate base pair average displacement (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002881#s4" target="_blank">Materials and Methods</a>). (C) Schematic affinity shape-correlation diagram of the examined <i>in vitro</i> sequences. Each point represents an oligomer with specific direct points-of-contact, correlation with the COP, and measured dissociation constant KD (the data is from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002881#pcbi.1002881.s001" target="_blank">Table S1</a>). The Pearson's correlation coefficient (horizontal axis) between the COP and the sequence shape and the KD (vertical logarithmic axis) are the (x, y) coordinates for each oligomer. The red circles depict sequences that violate at least two of the inclusion/exclusion rules while the blue squares correspond to the remaining sequences. The blue ellipse schematically depicts the majority of DNA sequences with good Fis-DNA binding (K_D< = 3 nM), and the other two ellipses schematically depict the majority of sequences with low affinity to Fis caused by bad point-of-contacts (pink) or low correlation with COP (green).</p